Periodontitis represents the most broadly distributed polymicrobial disease of mankind. The tissue destructive processes that lead to loss of function of periodontal soft tissues and alveolar bone result from a chronic polymicrobial biofilm activation of host inflammatory, innate immune, and adaptive immune responses in the local tissues. Development of the polymicrobial biofilm is a sequential process that begins with deposition of host and bacterial glycoproteins onto the clean tooth surface. Initial colonization by oral microorganisms occurs next, with oral streptococci being the primary early colonizers. This initial colonization by streptococci is followed by increasing proportions of Actinomyces, Veillonella, and Fusobacterium spp. (middle colonizers) with subsequent colonization predominated by Gram-negative, anaerobic bacteria (late colonizers). This shift from a predominance of Gram-positive, facultative microorganisms to a microbiota dominated by Gram- negative, anaerobic species is manifest clinically by inflammatory changes in the periodontal tissues as the host responds to this microbial challenge. It has been proposed that this multicellular bacterial-host interaction possesses unique features compared to planktonic bacterial infectious challenges;however, there is little objective evidence to support this contention. This R21 exploratory/development grant will specifically focus on developing this evidence using a novel polymicrobial biofilm model. The General Hypotheses to be tested are: (1) Oral bacteria in biofilms will elicit different patterns of host responses from gingival epithelial cells;and (2) Host cells will respond differently to bacterial species present as monospecies versus multispecies biofilms. We have developed a novel in vitro biofilm-host cell model system using a rigid, gas permeable contact lens material. In Specific Aim 1 we will determine the characteristics of single species and multispecies biofilms created on this material. The biofilms developed in this model system will then be used in Specific Aim 2 to challenge oral epithelial cells, allowing us to characterize the patterns of responses elicited in these host cells by the bacterial challenge. In accomplishing these aims, we will have developed a novel approach for evaluating host response patterns to planktonic, single species biofilm and polymicrobial biofilm challenges. The results obtained will enable us to provide seminal data to enable a further exploration of host responses to biofilms of various compositions and complexities. The bacteria found in dental plaque biofilms interact with tissues supporting the teeth to initiate an inflammatory response. The clinical diseases associated with this inflammatory response are gingivitis and/or periodontitis. However, the exact host responses elicited by this interaction have not been determined. We are developing an in vitro model of this interaction to gain a better understanding of the pathologenesis of gingivitis and periodontitis. This understanding is necessary to develop and evaluate new strategies for controlling the destructive features of these chronic inflammatory diseases.